Genus 1 Sp. 2 (Diptera: Chironomidae): the Potential Use of Its Larvae As Bioindicators

ISSN 2578-0336 Review Article Genus 1 sp. 2 (Diptera: Chironomidae): The Potential use of its Larvae as Bioindicators

Paula A Ossa López1, Narcís Prat2, Gabriel J Castaño Villa3, Erika M Ospina Pérez1, Ghennie T Rodriguez Rey4 and Fredy A Rivera Páez1* 1Grupo de Investigación GEBIOME, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia 2Grup de recerca consolidat F.E.M (Freshwater Ecology and Management), Departament de Biologia Evolutiva, Ecologia i Medi Ambient, Universitat de Barcelona, Barcelona, España 3Grupo de Investigación GEBIOME, Departamento de Desarrollo Rural y Recursos Naturales, Facultad de Ciencias Agropecuarias, Universidad de Caldas, Manizales, Caldas, Colombia 4Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Caldas, Colombia *Corresponding author: Fredy A Rivera Páez, Grupo de Investigación GEBIOME, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Caldas, Colombia

Submission: November 05, 2018; Published: November 16, 2018

Abstract The family Chironomidae belongs to the most abundant macroinvertebrates in samples for water quality assessment and displays a wide tolerance range to contaminants, which makes it an excellent bioindicator. The species Genus 1 sp. 2 (Chironomidae: Orthocladiinae), included among the larval

Chinchinákeys of the river Cricotopus-Oliveiriella basin (Caldas-Colombia), complex, along is difficultwith eight to organismsdetermine atbased the pupal on its level larval (reared instar in using the laboratory). the current The morphological organisms were keys, morphologically which makes it necessary to use pupae for a species-level identification. In this study, 103 organisms in the IV larval instar were collected from tributaries of the high

identified, and a molecular analysis of the genes COI and 16S rDNA was performed in order to confirm and associate larvae and pupae.

In the larval morphometric analysis, 13 structure measurements were taken, with the aim of finding possible variations among specimens from samplingdifferent samplingstations. Thestations, results and obtained only dorsal allow head for a areamolecular (DHAr) determination showed significant and association differences. of larvaeThe presence and pupae of mentumof the species deformities Genus 1was sp. assessed,2, and new a morphologicaltotal of 18 specimens measurements showed inpartial larvae or that total can teeth aid indeformity, determining although variations no significant resulting differencesfrom contaminant were found agents between and contributing deformity tofrequency establishing and thisthe species as a water quality bioindicator.

Keywords: Colombia; Deformities; Molecular analysis; Morphology; Morphometry

Introduction The subfamily Orthocladiinae (Diptera: Chironomidae) is one of species-level, the differences between species have not been studied the richest in genera and species, and in Andean rivers above 2000 complicating its identification even at the genus level, and at the yet. Its pupae, however, are very characteristic and very different meters of altitude, the subfamily Orthocladiinae is very abundant, from the genus Cricotopus; therefore, a species-level description can with multiple genera present in the high Andean region, and of be achieved. Larvae belonging to this taxon are abundantly found in which some have yet to be described [1]. Furthermore, species of the Chinchiná River (Colombia), and based on studies related to the the same genus share many larval characteristics, making it nearly association between macroinvertebrates and contamination, the impossible to distinguish them, even at a genus level. Nevertheless, possibility of using these larvae as contamination indicators has led to a complete morphological and genetic study in order to establish there are several records in which larvae and pupae have been pupal forms are specific for a genus, and even for a species, and its possible use as bioindicators [4,5]. associated in rivers of the high Andean region between Colombia and Peru. The use of aquatic macroinvertebrates currently constitutes a tool for the biological and integral characterization of water Among the most abundant genera of Orthocladiinae in the high quality [4,6]. All aquatic organisms can be considered as Andean region, there are larvae described as Genus 1 by Roback bioindicators, however, the evolutionary adaptations to different & Coffman [2], a genus found exclusively in the Andean region. environmental conditions and the tolerance limits to a given Its larvae belong to the Cricotopus-Oliveiriella complex [1,3]; disturbance are responsible for the characteristics that classify

Environ Anal Eco stud Copyright © Fredy A Rivera Páez them as sensitive organisms, whether they do not endure changes in their environment or they are tolerant to stress conditions. of 250µm, and manual drainers (the samples were taken from a Surber net, with 30.5x30.5x8cm dimensions and a mesh size Chironomidae (Diptera: Chironomidae), with nearly 20000 species sediments, rock washes and leaf litter). The specimens were distributed throughout all the continents, from the Antarctic region preserved in absolute ethanol with their corresponding information to the Tropics, inhabit lakes, streams and rivers during their larval (date, location, and coordinates). In the laboratory, several and pupal developmental stages [1,2,4]. specimens were conditioned in aquariums with water from the sampling stations, under constant oxygenation, and were fed with The family Chironomidae is considered to be tolerant to water TetraMin® contamination with organic matter, heavy metals, pesticides, aromatic polycyclic hydrocarbons, and organic solvents, displaying Additionally, until pupaethe werefollowing obtained physical for species and confirmation.hydrobiological subletal responses such as morphological variations represented variables were measured in situ in three of the six sampling events: by morphometric changes and deformities as a result of exposure to water temperature, pH, conductivity, dissolved oxygen, oxygen these conditions [5-11]. Regarding these tolerance characteristics, the following chemical variables were evaluated in the laboratory: saturation, average depth, width, and water flow velocity. Also, et al. [14,15], Giacometti & Bersosa [16], report Chironomidae chemical oxygen demand (COD), biological oxygen demand (BOD ), Arambourou et al. [11], Warwick [12], Alba-Tercedor [13], Servia 5 as organisms with a potential use in water quality bioindication. Nevertheless, one of the current limitations for the use of total coliforms, fecal coliforms, total suspended solids (TSS), total ammoniacal nitrogen (NH -N), phosphate (PO ), solids (TS), cyanide (CN), 3 boron (B), lead (Pb),4 mercury (Hg),4 which in many cases is not straightforward, due to phenotypic iron (Fe), chloride (Cl-), lipids and oils, nitrates (NO ), nitrites Chironomidae is an insufficient knowledge of their taxonomy, ), sulfate3 (SO plasticity or shared characters between several species of a genus, (NO2), and aluminum (Al). These variables were analyzed by or even between genera. chemical variables between the reference and mining stations were The present study aimed to morphological evaluation the ACUATEST S.A. Comparisons of the physical, hydrobiological, and performed using the Wilcoxon test (W). larvae of Genus 1 sp. 2 Roback & Coffman [2], through diagnostic characters, further, larvae and pupae of Genus 1 sp. 2 were Morphological and molecular evaluation molecularly determined and associated based on the study of The ethanol-preserved organisms were examined and mitochondrial genes. The possible morphometric variations and record the frequency of deformities was assessed in organisms of the stereomicroscope equipped with a MC170HD digital camera. Next, identified based on the keys of Prat et al. [1,3], using a Leica M205C the head of each specimen, both the larvae and pupae reared in impact or lack of any evident mining impact and sampling stations IV larval instar in the sampling stations (no evident anthropogenic the laboratory, were dissected and placed in hot 10% KOH. They with mining impact). Overall, the results allowing to contribute to were then washed, dehydrated, and mounted on microscope slides the establishment of this species as a water quality bioindicator. with Euparal® for their subsequent observation, following the light Materials and Methods microscopy (LM) techniques described by Epler [19], and the head capsule and pupal keys of Prat et al. [1,20]. Study area DNA was extracted from the thorax and abdomen of 11 larvae, The study area included six sampling stations located in the as well as two mature pupae, using the DNeasy Blood and Tissue Chinchiná River basin in the department of Caldas (Colombia). Kit (Qiagen®), according to the manufacturer’s instructions. Two sampling stations were selected as reference (areas without Two mitochondrial genes (mtDNA), cytochrome oxidase I (COI) an evident mining impact), one located in La Elvira stream (05°03’10.9’’ North, 75°24’33.6’’ West), municipality of Manizales, that were conducted following Ossa et al. [21]. The PCR products and the other in Romerales stream (04°59’22’’ North, 75°25’58’’ and 16S, were amplified with polymerase chain reactions (PCR) ía. The other four sampling stations according to the manufacturer’s instruction, and were shipped are impacted by waste disposal generated by gold mining [17,18]. were purified with the QIAquick PCR purification kit (Qiagen®), West), municipality of Villamar for sequencing at Macrogen Inc. Korea. The sequenced fragments Two of the stations were located in El Elvira stream, Manizales were evaluated and edited using Geneious Trial v8.14 [22] and (05°03’4.4’’ North, 75°24’33.1’’ West; 5°1’53’’ North, 75°24’43.8’’

(04°59’5’’ North, 75°26’35’’ West), and the last sampling station Sequencher 4.1 (Gene Codes Corporation, Ann Arbor, Michigan, West), another was located in California stream, Villamaría the public databases and deposited in GenBank and Barcode of Life was in Toldafrí ía (4°59’08’’ North, 75°26’43’’ USA). In addition, the sequences were search by MegaBlast against West). The six sampling stations stood between 2275 and 2766 a stream, Villamar meters of altitude, and had similar physical habitat characteristics, DataThere Systems are (BOLD) no available (Genbank sequences accessions for speciesKY568875-KY568909). of the Genus 1 such as a wavy topography and the presence of riparian vegetation in the public databases; therefore, the analysis of the mtDNA COI [17,18]. gene included sequences from eight species of Cricotopus, a genus with very similar larvae to Genus 1 [1]. The reason for including Specimen collection eight species from different subgenera of Cricotopus was to be able A total of six sampling events were conducted from February to more clearly establish the position of larvae of Genus 1 sp. 2 2014 to February 2015. Larvae collection was carried out with within the Cricotopus-Oliveiriella complex, since, similarly to what

Volume -4 Issue - 3 Paula A O L, Narcís P, Gabriel J C V, Erika M O P, Fredy A R P, et all. Genus 1 sp. 2 (Diptera: Chironomidae): The Potential use of How to cite this article: 377 its Larvae as Bioindicators. Environ Anal Eco stud. 4(3). EAES.000589. 2018. DOI: 10.31031/EAES.2018.04.000589

Environ Anal Eco stud Copyright © Fredy A Rivera Páez happened with the genus Oliveiriella, it is suspected that the larvae of Genus 1 of Roback are actually a subgenus within Cricotopus. performed using R version 3.1.1 (R Development Core Team 2011). non-parametric Wilcoxon test (W). Statistical analyses were Moreover, the species Limnophyes sp. was included as an In addition, several structures were re-assessed using an a species of Cricotopus and the species Cardiocladius sp. were used mounted on stubs and metalized in gold, then; the material was outgroup. For the mtDNA 16S rDNA gene analyses, sequences from electron scanning microscope (ESM). For this, the heads were as outgroups. The sequences for each gene were aligned using Clustal W [23], included in the program MEGA version 7 [24] and electron scanning microscope. The head capsule mounts with analyzed and photo-documented on a FEI QUANTA 250, ESEM the alignments were visually reviewed and edited when necessary. evaluate the possible existence of mouth deformities according to ML, as well as the ESM observations, were analyzed in order to the descriptions of Warwick [5] and Groenendijk et al. [28]. The program MEGA, using the Kimura 2-Parameter distance model Intraspecific nucleotide divergences were estimated with the association between deformity occurrence and the reference or (K P) [25]. Automatic Barcode Gap Discovery (ABGD; Puillandre et 2 mining stations was examined through Fisher’s Exact Test. al. [26] was used to infer the number of putative species, using an

2P Result intraspecific divergence prior ranging from 0.001 to 0.1 and the K Morphological and molecular evaluation a similarity analysis based on Neighbor-Joining (NJ), with the K P evolutionary model. Species confirmation was carried out through2 model and 1000 bootstrap replications, using the program MEGA. The morphological evaluation was based on the collection of 103 organisms of the morphotype Genus 1 sp. 2 (Table 1), Larval morphometric analyses and frequency of mentum deformities eight pupae reared in the laboratory. The larvae of Genus 1 are corresponding to the IV larval instar (Figure 1A), as well as morphologically characterized by a white colored body in young the reference and mining stations (lack of any evident mining larvae, and darker areas in the thorax in more mature larvae In order to find possible variations between specimens of impact and sampling stations with mining impact), 13 structure (Figure 1A). However, it has been noted that color variations cause measurements were recorded (mm or mm2 for areas), reported by Cranston & Krosch [27] for larvae of the genus Barbadocladius Genus 1 sp. 2 show equal abdominal setae of length corresponding difficulties for species determination [29]. Fourth instar larvae of (Diptera: Chironomidae) and other genera explored in this study, to half of the width of the abdominal segment, and anal tubules which were: lateral head length (LH), lateral head width (LHW), shorter than the posterior pseudopods (Figure 1B). Head with no lateral head length from the base (LHB), thorax length (TL), width pattern, very dark solid color with lighter areas close to the lateral border in the frontal and medial sections; very dark occipital total body length (TB), body area (BA), body perimeter (BP), dorsal border (Figures 1C-D) and short antennae (Figures 1D-E). Mentum of III thorax segment (WTS), width of IV abdominal segment (WAS), head length (DHL), dorsal head width (DHW), dorsal head area (DHAr), and dorsal head perimeter (DHP). The body measurements wide as second and narrower in the lower part (Figures 1C-E; 2A). with second lateral tooth smaller than the first; first lateral tooth as were compared between reference and mining sites, through the

Figure 1: Genus 1 sp. 2. IV larval instar. (A) Larva in IV instar (LM); (B) Abdominal termination; (C) Head cavity (ventral view); (D) Head cavity (dorsal view); (E) Head cavity. Mentum (M), Mandible (MA), Antenna (A). 1F-H. Genus 1 sp. 2; pupae. (F) Mount of IV abdominal segment (SA). Middle spines (SA III-VI), with hooklets (SA II-V); (G) Last abdominal segments and anal lobes; (H) Respiratory organs (OR) (Light microscopy-LM).

Volume -4 Issue - 3 How to cite this article: Paula A O L, Narcís P, Gabriel J C V, Erika M O P, Fredy A R P, et all. Genus 1 sp. 2 (Diptera: Chironomidae): The Potential use of 378 its Larvae as Bioindicators. Environ Anal Eco stud. 4(3). EAES.000589. 2018. DOI: 10.31031/EAES.2018.04.000589

Table 1: Larvae (IV instar) of Genus 1 sp. 2 with presence or absence of deformities in each sampling station.

Genus 1 sp. 2 Sampling Station With Deformities without Deformities Total E1 1 4 5

E4 2 6 8 E2 6 31 37 E3 4 27 31 E5 4 12 16 E6 1 5 6 Total 18 85 103 E1: La Elvira Stream (Reference Area); E2: La Elvira Stream (Mining); E3: La Elvira Stream (Mining), E4: Romerales Stream (Reference Area); E5: California Stream (Mining); E6: Toldafría stream (Mining) In addition, the molecular alignment analyses of the fragments (Figure 1E). In the eight pupae evaluated, terga ornamentation Mandible with upper tooth shorter and narrower than the first results obtained by the morphological determination. Based on of the mtDNA COI and 16S rDNA genes, respectively, confirmed the showed two rows of anteriorly-oriented spines in the II-V abdominal the genital sacs (Figure 1G). Respiratory organ (OR) rounded at the 0.001 and 0.1 for the COI gene and between 0.0028 and 0.0599 for segments (SA) (Figure 1F); anal lobe reduced, with a similar size to the initial partition and an intraspecific divergence prior between end and often with diverse folds on the surface (Figures 1H) and the larval and pupal sequences obtained for Genus 1 sp. 2 belong to the 16S gene, the ABGD species delimitation method identified that terga (Figure 1F). without middle spines in the II tergite, although present in the III-VI a single species out of the nine species identified with the COI gene and the two species identified with the 16S gene (Figure 2).

Figure 2: Genus 1 sp. 2. Head cavity, arrows indicate teeth deformities in the mentum. (A) Mentum without deformities-ESM; (B, E) Total loss of several dental pieces - ESM; (C) Teeth wear-LM; (D) Total loss of several dental pieces-LM (Light microscopy-LM and electron scanning microscopy-ESM).

Furthermore, the consensus trees obtained from the two genes, based on the Neighbor-Joining method, clearly show that the larval 2 are similar to the mean values found for the Cricotopus species 4). These intraspecific divergence values observed for Genus 1 sp. and pupal sequences of Genus 1 sp. 2 constitute a well-supported analyzed; with 0% for C. trifascia and 2.31% for C. bicinctus with

gene. monophyletic clade, with a mean intraspecific divergence of 0.95%, the COI gene, and 0.20% para Cricotopus (Oliveiriella) with the 16S based on the COI gene (Figure 3), and 0.11% on the 16S gene (Figure Volume -4 Issue - 3 Paula A O L, Narcís P, Gabriel J C V, Erika M O P, Fredy A R P, et all. Genus 1 sp. 2 (Diptera: Chironomidae): The Potential use of How to cite this article: 379 its Larvae as Bioindicators. Environ Anal Eco stud. 4(3). EAES.000589. 2018. DOI: 10.31031/EAES.2018.04.000589

the observed divergence between Genus 1 sp. 2 and Cricotopus analyzed varied between 4.73% and 19.2% with gene COI; while The intraspecific divergence values found between the species (Oliveiriella) with gene 16S is 7.49%.

Figure 3: Consensus NJ tree with samples of Genus 1 sp. 2, based on distances of the mtDNA COI gene. Bootstrap values are indicated only for nodes with support greater than 70%.

Figure 4: Consensus NJ tree with samples of Genus 1 sp. 2, based on distances of the mtDNA 16S gene. Bootstrap values are indicated only for nodes with support greater than 70%.

Volume -4 Issue - 3 How to cite this article: Paula A O L, Narcís P, Gabriel J C V, Erika M O P, Fredy A R P, et all. Genus 1 sp. 2 (Diptera: Chironomidae): The Potential use of 380 its Larvae as Bioindicators. Environ Anal Eco stud. 4(3). EAES.000589. 2018. DOI: 10.31031/EAES.2018.04.000589

Larval morphometric analyses and frequency of mentum stations (Fisher’s Exact Test, p=0.669). Of the total organisms deformities collected in the reference stations, 23.1% showed deformities (Table 2), indicating that these sampling stations have some type of anthropic impact, as evidenced by the physical, hydrobiological, differences were observed only for dorsal head area (DHAr) Of the 13 structure measurements assessed, significant and chemical analyses, where most of the parameters assessed do between the specimens found in the reference and mining stations, not show differences between the reference and mining stations. according to the non-parametric Wilcoxon test (W=382,5, p=0,04). Additionally, there were differences in organism abundance of Mentum deformities were observed in 18 of the 103 specimens Genus 1 sp. 2 in relation to the reference and mining stations (Table evaluated (Table 1; Figures 2A-D). Partial wear of the teeth was 3). The organisms from Genus 1 sp. 2 showed a greater abundance evident (Figure 2C), as well as total wear or tooth loss (Figures in stations with mining (n=90) compared to the reference stations (n=13). for deformity occurrence in relation to the reference and mining 2B; 2D-E). Nevertheless, no significant differences were found Table 2: Physical, hydrobiological, and chemical characteristics of the streams assessed. The values correspond to the mean values of the parameters measured in each sampling station (Reference stations E1 and E4. Mining impact stations E2, E3, E5 and E6).

Sampling Station Variables Parameter Measurement Units E1 E2 E3 E4 E5 E6 Water temperature 12.7 13.8 13.8 12.5 13.73 14.4 °C pH 7.64 7.8 7.89 7.2 7.35 7.25

Physical Conductivity 206 291 195 252.7 87.1 376.9

Dissolved oxygen mg/LµS 7.55 6.03 4.75 4.9 5.11 4.73 Oxygen saturation % 99.2 65.3 65.27 65.4 67.5 67.3

Average depth cm 10.7 10 13.8 16.5 19.11 10.9 Hydrobiological Width m 2.23 2.43 3.05 6.4 5.01 5.75 m/s 0.43 0.64 0.5 0.73 0.69 0.55

ChemicalWater flowoxygen velocity demand mg/L 23.3 20.3 106 32.67 91.7 59.7

Biological oxygen demand mg/L 3.21 3.21 10 3.21 6.17 3.21

Total coliforms UFC/100mL 2983 9257 341733 2286 3977 1.00E+05

Fecal coliforms UFC/100mL 540 873 2740 504 1546 636

Total suspended solids mg/L 34.17 306 1384 6.67 242.3 18.1

Total solids mg/L 110.7 395 1497,3 85.3 569 161.3 Cyanide mg/L 0.09 0.093 0.093 0.09 0.09 0.093 Boron mg/L 0.83 0.83 0.83 0.83 0.83 0.83

Chemical Lead mg/L 0.05 0.085 0.05 0.02 0.02 0.02 Mercury mg/L 0.33 0.33 0.34 0.33 0.33 0.33 Ammoniacal nitrogen mg/L 0.1 0.21 0.22 0.11 0.35 0.11 Phosphate mg/L 0.7 1.2 3.5 0.3 2.3 0.2 mg/L 21 56 103.3 7.67 45.7 19.3

SulfateIron mg/L 0.4 1.34 3.68 0.163 1.87 0.53 Chloride mg/L 2.5 2.9 9.2 3 7.3 2.5 Lipids and oils mg/L 0.5 0.9 0.63 0.5 0.5 0.5 Nitrates mg/L 1 1.1 1.4 1 1 1 Nitrites mg/L 0.07 0.3 0.72 0.07 0.34 0.07 Aluminum mg/L 0.11 0.6 8.02 0.1 2.3 0.15

E1-E3: La Elvira Streams; E4: Romerales Stream; E5: California Stream; E6: Toldafría Stream (Adapted from Ossa et al. [24]).

Volume -4 Issue - 3 Paula A O L, Narcís P, Gabriel J C V, Erika M O P, Fredy A R P, et all. Genus 1 sp. 2 (Diptera: Chironomidae): The Potential use of How to cite this article: 381 its Larvae as Bioindicators. Environ Anal Eco stud. 4(3). EAES.000589. 2018. DOI: 10.31031/EAES.2018.04.000589

Table 3: Wilcoxon Test comparing the parameters measured (medians are shown) between reference stations (E1 and E4) and mining impact stations (E2, E3, E5, and E6).

Sampling Station Wilcoxon Test Variables Parameter Reference Mining W-Value p-Value

Biological oxygen demand (BOD5) 3.21 3.21 45 0.22 Chemical oxygen demand (COD) 27.5 48.5 470.323 0.323 8 230 60* 0.027*

Total suspended solids (TSS) 106 500 61* 0.018* Nitrates (NO ) 1 1 51 0.085 Total solids (TS),3

Nitrites (NO2) 0.07 0.155 54 0.051

4) 14.5 47.5 63* 0.013*

SulfateIron (Fe) (SO 0.175 1.105 66* 0.003* Chloride (Cl-) 2.5 2.5 46.50.3 0.265

Chemical Phosphate (PO4) 0.35 0.95 48 0.265 Lipids and oils 0.5 0.5 42 0.346

Ammoniacal nitrogen (NH3-N) 0.1 0.1 40 0.734 Cyanide (CN) 0.1 0.1 36 1 Aluminum (Al) 0.095 0.225 55.5 0.071 Mercury (Hg) 0.003 0.003 40.5 0.696 Lead (Pb) 0.01 0.02 44 0.43 Boron (B) 0.8 0.8 36 1 Total coliforms 3370 4450 56 0.067 Fecal coliforms 14.25 1250 52 0.146 Oxygen saturation 70.8 63.25 32 0.743 Dissolved oxygen 5.205 4.585 32 0.7428 Físicas pH 7.5 7.615 45 0.425 Temperature 12.55 13.6 56.5 0.061 Conductivity 162 163.5 34 0.892 Average depth 13.499 13.167 34.5 0.925 Hydrobiological Width 3.825 3.4 37.5 0.925 0.553 0.576 41 0.682

*Differences Water flow velocity Discussion The morphological evaluation was based on 103 larvae and The molecular results confirmed the morphological eight pupae of Genus 1 sp. 2, and all morphological characteristics determination, previous studies have reported interspecific agree with those reported by Prat et al. [1]. Although, some of the distances for Diptera similar to those reported here; Shouche between 1% and 9% in three species of Diptera. Ekrem et al. [32] characteristics previously mentioned appear in many larval forms, & Patole [31] observed interspecific distances with gene 16S including the genus Cricotopus, of which several subgenera and family Chironomidae. However, this reference value for species morphotypes, along with the Genus 1, are included in keys of the reported interspecific divergences of 16.2% for gene COI in the Cricotopus-Oliveiriella complex. Therefore, it is almost impossible comprise specimens from the Holarctic region, and there are still to differentiate them at the species level based on a larval instar [30]. identification is not enough, given that these studies mainly few studies that analyze specimens from the Neotropics, including However, this morphology is associated with very different pupal members of the subfamily Orthocladiinae. forms, which allowed us to reach a species level determination, following the key of Prat et al. [20] and the indications of the original The comparison of the molecular data of Oliveiriella and description of pupae for Genus 1 sp. 2 in Roback & Coffman [2]. Prat

Genus 1 with other subgenera of Cricotopus confirms the findings in high Andean rivers, where these are very characteristic and very [33] in that Oliveiriella is a subgenus of Cricotopus and Genus 1 of et al. [20] report that it is common to find Genus 1 in pupae forms of Andersen et al. (2013), which are also confirmed by Prat et al. different from Cricotopus. Roback and Coffman is also a subgenera.

Volume -4 Issue - 3 How to cite this article: Paula A O L, Narcís P, Gabriel J C V, Erika M O P, Fredy A R P, et all. Genus 1 sp. 2 (Diptera: Chironomidae): The Potential use of 382 its Larvae as Bioindicators. Environ Anal Eco stud. 4(3). EAES.000589. 2018. DOI: 10.31031/EAES.2018.04.000589

Nevertheless, the larval measurements have been used to ecological and genetic responses of aquatic macroinvertebrates” differentiate larval instars and sexual dimorphism [34,35] pupal (Grant 569/2016). and exuviae stages [36], exposure to contaminant agents by References evaluating size variations in head parts [5,9-14,19,28,37] and morphometric variations in adults in different regional gradients 1. ín C, Rieradevall M. (2011) Guide for the recognition of the larvae of Chironomidae (Diptera) of the high Andean [38]. In this study, only dorsal head area (DHAr) is informative; riversPrat N, of AcostaEcuador R, and Villamar Peru. Key to the determination of the genres. therefore, more research is necessary in order to determine if the 2. differences found in this study are due to genetic variability, stress Peruvian Altiplano Expedition. Part II. Aquatic Diptera including type (essential and/or toxic substances), the structures studied montaneRoback SS, Diamesinae Coffman WP and (1983) Orthocladiinae Results of the (Chironomidae) Catherwood Bolivian- from or the morphometric data used, or to a combination of all of these variables [9,10,14,39,40]. 3. Venezuela. Proceedings of Natural Sciences of Philadelphia 135: 9-79. recognition of the larvae of Chironomidae (Diptera) of the High Andean riversPrat N, of AcostaEcuador R, and Villamarín Peru. Key C, for Rieradevall the determination M (2012) of the Guide main for larval the deformity frequency and the sampling stations. The presence of morphotypes. Although no significant differences were found between deformities in Chironomidae larval instars is considered to result 4. Zúñiga MC, Cardona W (2009) Bioindicators of water quality and from exposure of these organisms to diverse contaminant agents [9,39,40]. Due to their tolerance, Chironomidae are considered environmental Flow. In: Carvajal MY and Castro LM (eds.), Environmental excellent water quality bioindicators, since they have regulation 5. flow:Warwick Concepts, WF (1985) experiences Morphological and challenges. abnormalities Colombia, inpp. Chironomidae 167-196. (Diptera) larvae as measures of toxic stress in freshwater ecosystems: mechanisms for metals such as Cu, Ni, Zn, Cd, Pb, Hg, and Mn, and Indexing antennal deformities in Chironomus Meigen. Canadian Journal for which they employ a homeostatic control for the uptake of essential and toxic metals through metallothioneins [11,41-43] 6. of Fisheries and Aquatic Sciences 42(12): 1881-1914. consequently, allowing them to survive in contaminated conditions in chironomid larvae as indicators of poliution (pesticide) stress. [44]. NetherlandsMadden CP, Journal Suter PJ,of Aquatic Nicholson Ecology BC, Austin26(2-4): AD 551-557. (1992) Deformities

A great amount of total and suspended solids were found, with 7. Dickman M, Rygiel G (1996) Chironomid larval deformity frequencies, mortality and diversity in heavy-metal contaminated sediments of a a high content of sulfates and metals such as iron (Fe), which are Canadian riverine wetland. Environment International 22(6): 693-703. characteristic of mining disposals and can have negative effects on 8. Roldán G (1999) Macroinvertebrates and their value as indicators of exposed organisms [45]. Nevertheless, according to Arambourou water quality. Journal of the Colombian Academy of Exact Physical and et al. [11], there is missing information regarding the study of the

9. Natural Sciences 23: 375-387.ález MA (1999a) On the possible repercussion al. [40] mention that, to date, there are no studies that allow for of the presence of deformities in the life cycle of Chironomus riparius origin of these abnormalities. Servia et al. [14] and Arambourou et discarding the possibility that this type of malformations appear Servia MJ, Cobo F, Gonz Entomology Association 23: 105-113. spontaneously due to natural developmental defects. Further, it Meigen, 1804 (Diptera, Chironomidae). Bulletin of the Spanish cannot be ignored that changes in the mentum can be due to the 10. ález MA (1999b) Appearance of deformities in larvae of the genus Chironomus (Díptera, Chironomidae) collected substrate or contamination [39,46]. Servia MJ, Cobo F, Gonz Entomology 23: 331-332. Considering that genera of the order Diptera are typical of in unaltered environments. Bulletin of the Spanish Association of disturbed areas [4], Genus 1 sp. 2 can be considered as having 11. Arambourou H, Gismondi E, Branchu P, Beisel JN (2013) Biochemical potential for water quality bioindication, due to its tolerance and morphological responses in Chironomus riparius (Diptera, Chironomidae) larvae exposed to lead-spiked sediment. Environ Toxicol to environmental stress, similar to other species of the family Chem 32(11): 2558-2564. Chironomidae [5,9,10,16,40]. 12. Warwick WF (1990) The use of morphological deformities in chironomid Finally, the results obtained allow the molecular determination larvae for biological effects monitoring, Inland Waters Directorate, National Hydrology Research Institute, National Hydrology Research of Genus 1 sp. 2 [2], support the morphological data, and associate Centre, Environment Canada. larvae and pupae, contributing to a better understanding of the 13. Alba TJ (1996) Aquatic macroinvertebrates and water quality of rivers, í 213. taxonomical limits in Chironomidae, specifically the subfamily determination of its species [30,44,47-49]. Moreover, the results in IV Water Symposium in Andalusia (SIAGA) - Almer a II, Spain pp. 203- Orthocladiinae, where there are many difficulties in the taxonomic 14. á í support the establishment of this species as a water quality variations in me frequency and severity of deformities in larvae of bioindicator [50-52]. ChironomusServia MJ, Cobo riparias F, GonzMeigen,lez 1804 MA (2000a)and Prodiamesa Seasonal oliv andá cea interannua (Meigen, 1818) (Díptera, Chironomidae) collecled in apolluted site. Environmental Acknowledgment Monitoring and Assessment 64: 617-626. To the members of the Research Group GEBIOME, the Institute 15. ález MA (2000b) Incidence and causes of deformities in recently hatched larvae of Chironomus riparius Meigen, 1804Servia (D MJ,íptera, Cobo Chironomidae). F, Gonz Archiv für Hydrobiologie 349: 387-401. Collection of the Biology Program of the Universidad de Caldas IIES, the Laboratory of Microbiology, and the Entomological 16. their importance as bioindicators of water quality in the Alambi River of impacts of mining, agriculture and livestock farming through Giacometti JCV, Bersosa FV (2006) Aquatic macroinvertebrates and (CEBUC). To COLCIENCIAS for financing the project “Assessment Technical Bulletin 6. Zoological Series 2:17-32. Volume -4 Issue - 3 Paula A O L, Narcís P, Gabriel J C V, Erika M O P, Fredy A R P, et all. Genus 1 sp. 2 (Diptera: Chironomidae): The Potential use of How to cite this article: 383 its Larvae as Bioindicators. Environ Anal Eco stud. 4(3). EAES.000589. 2018. DOI: 10.31031/EAES.2018.04.000589

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Volume -4 Issue - 3 How to cite this article: Paula A O L, Narcís P, Gabriel J C V, Erika M O P, Fredy A R P, et all. Genus 1 sp. 2 (Diptera: Chironomidae): The Potential use of 384 its Larvae as Bioindicators. Environ Anal Eco stud. 4(3). EAES.000589. 2018. DOI: 10.31031/EAES.2018.04.000589

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Volume -4 Issue - 3 Paula A O L, Narcís P, Gabriel J C V, Erika M O P, Fredy A R P, et all. Genus 1 sp. 2 (Diptera: Chironomidae): The Potential use of How to cite this article: 385 its Larvae as Bioindicators. Environ Anal Eco stud. 4(3). EAES.000589. 2018. DOI: 10.31031/EAES.2018.04.000589